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Microdetermination of α-amino acids by spectrophotometric and titrimetric methods
Talanta,
1971, Vol. 19, pp. 31 to 36. Pcrgamon
Press.
Pnnred tn Nonhem
hiand
MICRODETERMINATION OF x-AMINO ACIDS BY SPECTROPHOTOMETRIC AND TITRIMETRIC METHODS W. I. AWAD Department
Department
of Chemistry, S. NASHED, of Chemistry,
University College for U.A.R.
Women, Ain Shams University, Cairo
S. S. M. HASSAN and R. F. ZAKHARY Faculty of Science, Ain Shams University,
Cairo, U.A.R.
(Received 16 April 1971. Accepted 22 June 1971) Summary-Micro-methods for spectrophotometric and oxidimetric determination of a-amino-acids are described. They are based on a reaction of the acids with ~er~-naphth~d~-Z,3,4-t~one hydrate at pH 2.5 to give a red precipitate of dihydroxy-fern-naph~ind~o~e. The red product is dissolved and measured either spectrophotometrically at 243 nm or titrimetrically by reaction with iodine or Nbromosuccinimide. The results obtained by the three methods are concordant, the average recovery being 98 %.
MANY METHODS have been reported for the determination of z-amino-acids. Alkalimetric,’ acidimetric,2 manometric,3 gasometric,q and chromatographic5 measurements of nitrous oxide, nitrogen, ammonia, carbon dioxide and aldehydes produced by reactions with suitable reagents have been proposed. Direct alkalimetric and complexometric ’ determination of the free acid after treatment with excess uf formaldehyde and copper salts are employed. Enzymatic,8 mass spectrometrics and conductometricr” methods have been also utilized. However, few calorimetric and redox reactions are known for amino-acid determination. Calorimetric reactions with ninhydrin, I1 3,5dibromosalicyldehydel’ and ~diacetyl~nzene I3 have been mentioned. A method based on a reaction of Nethylmaleimide with carboxydithiocarbamate, formed by the action of carbon disulphide on the acid, is described. l3 An oxidimetric method based on deamination of the acid followed by oxidation of the resulting hydroxy-acid with vanadium(V) has been used.15 fern-napht~ndan-2,3,4-trione hydrate has been proposed for the determination of a-amino-acids by measuring the amount of carbon dioxide,r6 ammonia,l’ and aldehydeP produced. In the present work this reagent is used in the development of new calorimetric and redox methods for amino-acid determination. EXPERIMENTAL Reagents All reagents were of analytical grade unless otherwise specified. Peri-naphthindan-2,3,4-trione hydrate was prepared as described previously.lB*ls ~ihydroxy-~~-~phIh~nden~n~ was prepared by reduction of the trione with ascorbic acid.‘O Citrate buffer solution. Sodium citrate trihydrate (2.06 g) and 19.16 g of citric acid monohydrate were dissolved in 500 ml of water. The a-amino-acids used were of purity not less than 99%. Procedure ~pec~rophuf#metric deferm~nafj~n ofu-amino-acids. Construct a standard caiibration curve as fohows. Dissolve l-765 mg of dihydroxy-peri-naphthindenone in 50 ml of methanol. Then take 15, 31
W. I.
32
S.
AWAD,
NASHED,
S. S. M. HA~X
and R. F.
ZAKHARY
7 ‘.5 , 3,3.5,4.4.5 and 5 ml of this solution in lo-ml measuring flasks and make up to the mark with -,methanol. Measure the absorbance of these solutions at 3-Q nm in lo-mm cuvettes within 15 min, usilg methanol in the reference cell. Draw a graph of the absorbance against the concentratlon of amino-acid nitrogen (1 !cg of dihydroxy-peri-naphthindenone zzz0.066 pg of amino-acid nitrogen). Into a 25-ml round bottom flask, introduce 4 ml of 0.1 %peri-naphthindan-2,3,4-trione hydrate soiution in distilled water and 1 ml of the citrate buffer solution. Add 0.4 ml of 8m’M a-amino-acid solution in water (equivalent to a.8 pg of amino-acid nitrogen). Heat the ffask under reflux for I hr at loo”, cool in ice and fiiter ~hat~n No. 42 paper). Wash several times with cold water. DissoIve the red precipitate of ~hydro~y-berg-naphth~denone in methanoI into a 50-ml graduated flask. Measure the absorbance at 342 nm in a IO-mm cell against a blank. Titrimetric determination of x-amino-acrds with iodine. Dissolve the dihydroxy-peri-naphthindenone precipitate in 25 ml of methanol in a 250~ml conical flask. Add 100 ml of water and 5 ml of 0.004Niodine. After 5 min titrate the excess of iodine with 0.004Nsodium thiosulphate, using starch as indicator (1 ml of 0.004N iodine = 21 flueof amino-acid nitrogen). Titrimetric determination of st-amirto-ait with N-bromosnccin;mide. Dissolve the red precipitate in 50 ml of 1: 1 acetic acid-water in a 250~ml conica flask. Dilute with water to 150 ml, add 5 ml of 4:; potassium iodide solution and 1 ml of 1% starch solution and titrate with O-004N N-bromosuccinimide (I ml of 0.004N N-bromosuccinimide = 21 pg of amino-acid nitrogen).
RESULTS
AND
DISCUSSION
Peri-naphthindan-2,3,4-trione hydrate (I) decomposes u-amino-acids quantitatively with the formation of the corresponding aldehyde having one carbon atom less, ammonia, carbon dioxide and dihydroxy-peri-naphthindenone (II) according to the equation :
+
R-CH-COOH
+ R.CHO + NH, + CO>
-*
NH, (c&u~ess,
water-soluble) (I)
(Ey,
water-insoluble) (Ii)
Since one molecule of dihydroxy-peri-naphthindenone is produced for each molecule of a-amino-acid, this reaction may be used for amino-acid determination. The reaction is quantitative in a citrate buffer solution at pH 2-5 and a low blank value is obtained.
Absorptive spectra. The absorption spectra show maxima at 320 nm (E f-22 x IQ3 l.mole-l.mm-l) for the naphthindantrione and 342 nm (E 1.14 x IO3l.mole-l.mm-l) for the dihydroxynaphthindenone (Fig. 1). Because of the equal absorption by both compounds over a wide wavelength range, prior separation is necessary; in that case an excess of the reagent does riot adversely affect the accuracy of the method. Beer’s Iaw is obeyed (for dihydroxy-peri-naphthindenone) in the concentration range equivalent to 0.1-2 ,ug of amino-acid nitrogen per ml and only one calibration curve is required for all z-amino-acids. Efict ofpH. Dihydroxy-Peru-naphthindenone dissolves in methanol to give a red
,Microdetermination
0
33
of a-amino-acids
J
!Il3ljl/i"
300
320
343 Wavefength,
FE.
360
380
400
nm
1
0
300
320
360
340 Wavelsngth,
FIG.
380
400
nm
2
solution and in glacial acetic acid to give a yellow one. Both solutions absorb at around 340 nm with equal molar absorptivity, indicating that the colour intensity at this wavelength is independent of the acidity. Eflect of standing time. The absorbance of methanolic solutions containing different concentrations (up to 12 &ml) of dihydroxy-peri-naphthindenone was measured after 1,30 and 120 min and 24 hr. It was observed that the colour intensity
W. I. AWAD, S. NASHED, S. S. M. HAssAN and R. F. ZAKHARY
34
0
I
!
300
I 320
I
I
II
340
I 360
Wavelength,
I
I
380
I 400
nm
FIG. 3.
remained stable for 30 min, after which fading took place. On standing for 2 hr the intensity decreased by 5-10%. After 24 hr the dihydroxy-peri-naphthindenone changed (probably by air-oxidation) to peri-naphthindan-2,3,4-trione and the absorption maximum was shifted to 320 nm (Fig. 2). However, the dihydroxy compound was more stable in glacial acetic acid medium and SS% of the colour intensity remained after 24 hr (Fig. 3). Determination of u-amino-acids. A number of x-amino-acids were analysed for amino-nitrogen content. The results (Table I) show an average recovery of 98.2% and a mean absolute error of ho-3 %. Amino-acids containing thiol groups (e.g., cysteine hydrochloride) give high recoveries probably because of the interference of the thiol group in the reduction reaction.
TABLE I.-MICRODETERMINAT~ONOF Q-AMWO-ACIDS BY REACTIONWITH peri-NAPHTHINDAN-2,3,4-TRIONEHYDRATE Amino-acid Sample
Spectrophotometric Giycine Phenylalanine Glutamic acid monohydrate Aspartic acid Leucine
nitrogen,
‘A
Theory
18.65 8.48 8-48 10.52
Methionine
lo-67 9.38
Threonine Setine
11.75 13.32
Titrimetric (iodine)
19.9
18.7
8-6
8.5
8.4
8.4 10.1 10.3 9.2
10,2 10.3 9.2 11.3 12.8
11.5 12.7
Titrimetric (N-bromosuccinimide) 18.7 8.5 8.4 10.2 10.2 9.2 11.1 12.8
Microdetetination
of x-amino-acids
35
Titrimetric methods Reaction with N-bromosuccinimide. N-Bromosuccinimide quantitatively oxidizes the ene-diol group in dihydroxy-peri-naphthindenone to give peri-naphthindan-2,3,4trione. The reaction proceeds according to the equation:
For determining x-amino-acids, the equivalent amount of the dihydroxy compound is separated from the reaction medium by filtration, dissolved in acetic acid and titrated directly with standard N-bromosuccinimide solution. Reaction with iodine. The ene-diol group of dihydroxy-peri-naphthindenone reacts with iodine quantitatively according to the equation:
Excess of iodine solution is added to the dihydroxy compound (itself equivalent to the amino-acid) and the iodine remaining after reaction is titrated with sodium thiosulphate. Determination of x-amino-acids. Many samples of u-amino-acids were analysed by oxidimetric titration of the equivalent amount of dihydroxy-peri-naphthindenone obtained in the reaction with the trione. With N-bromosuccinimide, the results obtained show an average recovery of 97.8 % and a mean absolute error of &IO*3%. With iodine, the mean absolute error is &O-3%, the average recovery being 98.4% (Table 1). Zusammenfassung-Mikromethoden zur spektrophotometrischen und oxidimetrischen Bestimmung von a-Aminosiuren werden beschrieben. Sie beruhen auf der Reaktion der SIuren mit peri-Naphthindan-2,3,4trion-Hydrat be.i pH 2.5; es entsteht ein roter Niederschlag von Dihydroxy-peri-naphthindenon. Das rote Produkt wird Eel&t und entweder hpektrop-hotometrisch bei 243 nm oder titrimet&ch durch Reaktion mit Jod oder N-Bromsuccinimid bestimmt. Die Ergebnisse der drei Methoden stimmen iiberein, im Durchschnitt bet&t die Ausbeute 98 %. R&me-On d&it des micromtthodes pour la determination spectrophotomttrique et oxydimCtrique des a-amino acides. Elles sont basees sur une r&action des acides avec l’hydrate de peri-naphtindan2,3,4-trione a pH 2,5, donnant un pr6cipitb rouge de dihydroxy-perinaphtindtone. Le produit rouge est dissous et mesure, soit
36
W. I. Away, S. NASHED,S. S. M. H-AN
and R. F.
ZA~MY
spectrophotometriquement B 243 nm, soit titrimitriquement par reaction avec I’iode ou le N-bromosuccinimde. Les rbultats obtenus par les trois methodes sent concordants, la recuperation moyenne &ant de 98 %.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
REFERENCES D. van Slyke. D. MacFadyer and P. Hamilton, J. Bial. Chem., 1941, 141, 671. A. Patchornik and Y. Shalitin, Anal. Chem., 1961,33,1887. D. van Slyke, R. Dillon, D. MacFadyer and P. Hamilton, J. Biol. Chem., 1941. 141,627. M. Renard and P. Des&s, Mikrachim. Acfa, 1951,37,665. M. Bier and P. Teitelbaum, Ann. N. Y. Acad. Sci., 1959,72,64i. W. Taylor, Analyst, 1957,82,488. B. Gauthier and M. Marechal, Ann. Pharm. Franc. 1962, 20, 156. I-L Mahnstadt and T. Hadjiioannou, Anal. Chem. 1963,35, 14. G. Junk and H. Svec, Anal. Chim. Acta, 1963,28, 164. M. Tmkova and M. Voldan, Cesk. Farm., 1963,12,182. S. Moore and W. Stein, f. Biol. Chem., 1954,211,907. K. Y&i, J. Pharm. Sot. (Japan), 1961,81,297. R. Riemschneider and J. Wierer, 2. Anal, Chem., 1963,193, 186. Z. Peizker, Collection Czech. Chem. Commun., 1960.25, 1514. S. Rao, H. Rathi and J. Gau, Anal. Chim. Acta, 1961,25,136. R. Moubasher and A. Sina, J. Bial. Chem., 1949,180,681. R. Moubasher, W. I. Awad and A. Othman, ibid., 1950, 183,693. R. Moubasher and W. I. Awad, ibid., 1949,179,915. G.Errera, Gazr. Chim. Ital., 1913,43,593; 1914,44,18. R. Moubasher, J. Biol. Chem., 1948, 176, 529.
1971, Vol. 19, pp. 31 to 36. Pcrgamon
Press.
Pnnred tn Nonhem
hiand
MICRODETERMINATION OF x-AMINO ACIDS BY SPECTROPHOTOMETRIC AND TITRIMETRIC METHODS W. I. AWAD Department
Department
of Chemistry, S. NASHED, of Chemistry,
University College for U.A.R.
Women, Ain Shams University, Cairo
S. S. M. HASSAN and R. F. ZAKHARY Faculty of Science, Ain Shams University,
Cairo, U.A.R.
(Received 16 April 1971. Accepted 22 June 1971) Summary-Micro-methods for spectrophotometric and oxidimetric determination of a-amino-acids are described. They are based on a reaction of the acids with ~er~-naphth~d~-Z,3,4-t~one hydrate at pH 2.5 to give a red precipitate of dihydroxy-fern-naph~ind~o~e. The red product is dissolved and measured either spectrophotometrically at 243 nm or titrimetrically by reaction with iodine or Nbromosuccinimide. The results obtained by the three methods are concordant, the average recovery being 98 %.
MANY METHODS have been reported for the determination of z-amino-acids. Alkalimetric,’ acidimetric,2 manometric,3 gasometric,q and chromatographic5 measurements of nitrous oxide, nitrogen, ammonia, carbon dioxide and aldehydes produced by reactions with suitable reagents have been proposed. Direct alkalimetric and complexometric ’ determination of the free acid after treatment with excess uf formaldehyde and copper salts are employed. Enzymatic,8 mass spectrometrics and conductometricr” methods have been also utilized. However, few calorimetric and redox reactions are known for amino-acid determination. Calorimetric reactions with ninhydrin, I1 3,5dibromosalicyldehydel’ and ~diacetyl~nzene I3 have been mentioned. A method based on a reaction of Nethylmaleimide with carboxydithiocarbamate, formed by the action of carbon disulphide on the acid, is described. l3 An oxidimetric method based on deamination of the acid followed by oxidation of the resulting hydroxy-acid with vanadium(V) has been used.15 fern-napht~ndan-2,3,4-trione hydrate has been proposed for the determination of a-amino-acids by measuring the amount of carbon dioxide,r6 ammonia,l’ and aldehydeP produced. In the present work this reagent is used in the development of new calorimetric and redox methods for amino-acid determination. EXPERIMENTAL Reagents All reagents were of analytical grade unless otherwise specified. Peri-naphthindan-2,3,4-trione hydrate was prepared as described previously.lB*ls ~ihydroxy-~~-~phIh~nden~n~ was prepared by reduction of the trione with ascorbic acid.‘O Citrate buffer solution. Sodium citrate trihydrate (2.06 g) and 19.16 g of citric acid monohydrate were dissolved in 500 ml of water. The a-amino-acids used were of purity not less than 99%. Procedure ~pec~rophuf#metric deferm~nafj~n ofu-amino-acids. Construct a standard caiibration curve as fohows. Dissolve l-765 mg of dihydroxy-peri-naphthindenone in 50 ml of methanol. Then take 15, 31
W. I.
32
S.
AWAD,
NASHED,
S. S. M. HA~X
and R. F.
ZAKHARY
7 ‘.5 , 3,3.5,4.4.5 and 5 ml of this solution in lo-ml measuring flasks and make up to the mark with -,methanol. Measure the absorbance of these solutions at 3-Q nm in lo-mm cuvettes within 15 min, usilg methanol in the reference cell. Draw a graph of the absorbance against the concentratlon of amino-acid nitrogen (1 !cg of dihydroxy-peri-naphthindenone zzz0.066 pg of amino-acid nitrogen). Into a 25-ml round bottom flask, introduce 4 ml of 0.1 %peri-naphthindan-2,3,4-trione hydrate soiution in distilled water and 1 ml of the citrate buffer solution. Add 0.4 ml of 8m’M a-amino-acid solution in water (equivalent to a.8 pg of amino-acid nitrogen). Heat the ffask under reflux for I hr at loo”, cool in ice and fiiter ~hat~n No. 42 paper). Wash several times with cold water. DissoIve the red precipitate of ~hydro~y-berg-naphth~denone in methanoI into a 50-ml graduated flask. Measure the absorbance at 342 nm in a IO-mm cell against a blank. Titrimetric determination of x-amino-acrds with iodine. Dissolve the dihydroxy-peri-naphthindenone precipitate in 25 ml of methanol in a 250~ml conical flask. Add 100 ml of water and 5 ml of 0.004Niodine. After 5 min titrate the excess of iodine with 0.004Nsodium thiosulphate, using starch as indicator (1 ml of 0.004N iodine = 21 flueof amino-acid nitrogen). Titrimetric determination of st-amirto-ait with N-bromosnccin;mide. Dissolve the red precipitate in 50 ml of 1: 1 acetic acid-water in a 250~ml conica flask. Dilute with water to 150 ml, add 5 ml of 4:; potassium iodide solution and 1 ml of 1% starch solution and titrate with O-004N N-bromosuccinimide (I ml of 0.004N N-bromosuccinimide = 21 pg of amino-acid nitrogen).
RESULTS
AND
DISCUSSION
Peri-naphthindan-2,3,4-trione hydrate (I) decomposes u-amino-acids quantitatively with the formation of the corresponding aldehyde having one carbon atom less, ammonia, carbon dioxide and dihydroxy-peri-naphthindenone (II) according to the equation :
+
R-CH-COOH
+ R.CHO + NH, + CO>
-*
NH, (c&u~ess,
water-soluble) (I)
(Ey,
water-insoluble) (Ii)
Since one molecule of dihydroxy-peri-naphthindenone is produced for each molecule of a-amino-acid, this reaction may be used for amino-acid determination. The reaction is quantitative in a citrate buffer solution at pH 2-5 and a low blank value is obtained.
Absorptive spectra. The absorption spectra show maxima at 320 nm (E f-22 x IQ3 l.mole-l.mm-l) for the naphthindantrione and 342 nm (E 1.14 x IO3l.mole-l.mm-l) for the dihydroxynaphthindenone (Fig. 1). Because of the equal absorption by both compounds over a wide wavelength range, prior separation is necessary; in that case an excess of the reagent does riot adversely affect the accuracy of the method. Beer’s Iaw is obeyed (for dihydroxy-peri-naphthindenone) in the concentration range equivalent to 0.1-2 ,ug of amino-acid nitrogen per ml and only one calibration curve is required for all z-amino-acids. Efict ofpH. Dihydroxy-Peru-naphthindenone dissolves in methanol to give a red
,Microdetermination
0
33
of a-amino-acids
J
!Il3ljl/i"
300
320
343 Wavefength,
FE.
360
380
400
nm
1
0
300
320
360
340 Wavelsngth,
FIG.
380
400
nm
2
solution and in glacial acetic acid to give a yellow one. Both solutions absorb at around 340 nm with equal molar absorptivity, indicating that the colour intensity at this wavelength is independent of the acidity. Eflect of standing time. The absorbance of methanolic solutions containing different concentrations (up to 12 &ml) of dihydroxy-peri-naphthindenone was measured after 1,30 and 120 min and 24 hr. It was observed that the colour intensity
W. I. AWAD, S. NASHED, S. S. M. HAssAN and R. F. ZAKHARY
34
0
I
!
300
I 320
I
I
II
340
I 360
Wavelength,
I
I
380
I 400
nm
FIG. 3.
remained stable for 30 min, after which fading took place. On standing for 2 hr the intensity decreased by 5-10%. After 24 hr the dihydroxy-peri-naphthindenone changed (probably by air-oxidation) to peri-naphthindan-2,3,4-trione and the absorption maximum was shifted to 320 nm (Fig. 2). However, the dihydroxy compound was more stable in glacial acetic acid medium and SS% of the colour intensity remained after 24 hr (Fig. 3). Determination of u-amino-acids. A number of x-amino-acids were analysed for amino-nitrogen content. The results (Table I) show an average recovery of 98.2% and a mean absolute error of ho-3 %. Amino-acids containing thiol groups (e.g., cysteine hydrochloride) give high recoveries probably because of the interference of the thiol group in the reduction reaction.
TABLE I.-MICRODETERMINAT~ONOF Q-AMWO-ACIDS BY REACTIONWITH peri-NAPHTHINDAN-2,3,4-TRIONEHYDRATE Amino-acid Sample
Spectrophotometric Giycine Phenylalanine Glutamic acid monohydrate Aspartic acid Leucine
nitrogen,
‘A
Theory
18.65 8.48 8-48 10.52
Methionine
lo-67 9.38
Threonine Setine
11.75 13.32
Titrimetric (iodine)
19.9
18.7
8-6
8.5
8.4
8.4 10.1 10.3 9.2
10,2 10.3 9.2 11.3 12.8
11.5 12.7
Titrimetric (N-bromosuccinimide) 18.7 8.5 8.4 10.2 10.2 9.2 11.1 12.8
Microdetetination
of x-amino-acids
35
Titrimetric methods Reaction with N-bromosuccinimide. N-Bromosuccinimide quantitatively oxidizes the ene-diol group in dihydroxy-peri-naphthindenone to give peri-naphthindan-2,3,4trione. The reaction proceeds according to the equation:
For determining x-amino-acids, the equivalent amount of the dihydroxy compound is separated from the reaction medium by filtration, dissolved in acetic acid and titrated directly with standard N-bromosuccinimide solution. Reaction with iodine. The ene-diol group of dihydroxy-peri-naphthindenone reacts with iodine quantitatively according to the equation:
Excess of iodine solution is added to the dihydroxy compound (itself equivalent to the amino-acid) and the iodine remaining after reaction is titrated with sodium thiosulphate. Determination of x-amino-acids. Many samples of u-amino-acids were analysed by oxidimetric titration of the equivalent amount of dihydroxy-peri-naphthindenone obtained in the reaction with the trione. With N-bromosuccinimide, the results obtained show an average recovery of 97.8 % and a mean absolute error of &IO*3%. With iodine, the mean absolute error is &O-3%, the average recovery being 98.4% (Table 1). Zusammenfassung-Mikromethoden zur spektrophotometrischen und oxidimetrischen Bestimmung von a-Aminosiuren werden beschrieben. Sie beruhen auf der Reaktion der SIuren mit peri-Naphthindan-2,3,4trion-Hydrat be.i pH 2.5; es entsteht ein roter Niederschlag von Dihydroxy-peri-naphthindenon. Das rote Produkt wird Eel&t und entweder hpektrop-hotometrisch bei 243 nm oder titrimet&ch durch Reaktion mit Jod oder N-Bromsuccinimid bestimmt. Die Ergebnisse der drei Methoden stimmen iiberein, im Durchschnitt bet&t die Ausbeute 98 %. R&me-On d&it des micromtthodes pour la determination spectrophotomttrique et oxydimCtrique des a-amino acides. Elles sont basees sur une r&action des acides avec l’hydrate de peri-naphtindan2,3,4-trione a pH 2,5, donnant un pr6cipitb rouge de dihydroxy-perinaphtindtone. Le produit rouge est dissous et mesure, soit
36
W. I. Away, S. NASHED,S. S. M. H-AN
and R. F.
ZA~MY
spectrophotometriquement B 243 nm, soit titrimitriquement par reaction avec I’iode ou le N-bromosuccinimde. Les rbultats obtenus par les trois methodes sent concordants, la recuperation moyenne &ant de 98 %.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
REFERENCES D. van Slyke. D. MacFadyer and P. Hamilton, J. Bial. Chem., 1941, 141, 671. A. Patchornik and Y. Shalitin, Anal. Chem., 1961,33,1887. D. van Slyke, R. Dillon, D. MacFadyer and P. Hamilton, J. Biol. Chem., 1941. 141,627. M. Renard and P. Des&s, Mikrachim. Acfa, 1951,37,665. M. Bier and P. Teitelbaum, Ann. N. Y. Acad. Sci., 1959,72,64i. W. Taylor, Analyst, 1957,82,488. B. Gauthier and M. Marechal, Ann. Pharm. Franc. 1962, 20, 156. I-L Mahnstadt and T. Hadjiioannou, Anal. Chem. 1963,35, 14. G. Junk and H. Svec, Anal. Chim. Acta, 1963,28, 164. M. Tmkova and M. Voldan, Cesk. Farm., 1963,12,182. S. Moore and W. Stein, f. Biol. Chem., 1954,211,907. K. Y&i, J. Pharm. Sot. (Japan), 1961,81,297. R. Riemschneider and J. Wierer, 2. Anal, Chem., 1963,193, 186. Z. Peizker, Collection Czech. Chem. Commun., 1960.25, 1514. S. Rao, H. Rathi and J. Gau, Anal. Chim. Acta, 1961,25,136. R. Moubasher and A. Sina, J. Bial. Chem., 1949,180,681. R. Moubasher, W. I. Awad and A. Othman, ibid., 1950, 183,693. R. Moubasher and W. I. Awad, ibid., 1949,179,915. G.Errera, Gazr. Chim. Ital., 1913,43,593; 1914,44,18. R. Moubasher, J. Biol. Chem., 1948, 176, 529.